title: Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs.
abstract: BACKGROUND
  Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses.
  
  
  METHODS
  C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs.
  
  
  RESULTS
  In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy.
  
  
  CONCLUSIONS
  Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.
authors.affiliation: Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX, USA. bigong@utmb.edu
authors.forename: Bin
authors.initials: B
authors.lastname: Gong
authors.forename: Yong Sun
authors.initials: YS
authors.lastname: Lee
authors.forename: Inhan
authors.initials: I
authors.lastname: Lee
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Shelite
authors.forename: Nawapol
authors.initials: N
authors.lastname: Kunkeaw
authors.forename: Guang
authors.initials: G
authors.lastname: Xu
authors.forename: Kwanbok
authors.initials: K
authors.lastname: Lee
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Jeon
authors.forename: Betty H
authors.initials: BH
authors.lastname: Johnson
authors.forename: Qing
authors.initials: Q
authors.lastname: Chang
authors.forename: Tuha
authors.initials: T
authors.lastname: Ha
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Mendell
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cheng
authors.forename: Donald H
authors.initials: DH
authors.lastname: Bouyer
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Boor
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Ksiazek
authors.forename: David H
authors.initials: DH
authors.lastname: Walker
mesh_terms: D000818:Animals; D001483:Base Sequence; D001907:Boutonneuse Fever / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D042783:Endothelial Cells / Q000378:metabolism / Q000382:microbiology / Q000473:pathology*; D004730:Endothelium, Vascular / Q000378:metabolism / Q000382:microbiology; D005260:Female; D054884:Host-Pathogen Interactions; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D058727:RNA, Small Untranslated / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*; D020114:Rickettsia conorii / Q000472:pathogenicity / Q000502:physiology*; D015854:Up-Regulation
chemical_list: D058727:RNA, Small Untranslated; D011994:Recombinant Proteins; D012343:RNA, Transfer; C046366:angiogenin; D012259:Ribonuclease, Pancreatic